*********A********* By Authority Of THE UNITED STATES OF AMERICA Legally Binding Document By the Authority Vested By Part 5 of the United States Code § 552(a) and Part 1 of the Code of Regulations § 51 the attached document has been duly INCORPORATED BY REFERENCE and shall be considered legally binding upon all citizens and residents of the United States of America. HEED THIS NOTICE : Criminal penalties may apply for noncompliance. Document Name: APHA Method 9215: Standard Methods for the Examination of Water and Wastewater CFR Section(s) : 40 CFR 141121 Standards Body: American Public Health Association Standard For the Examination of Water and Wastewater : mm Imm^mmmm^mmm^ Prepared and published jointly by: American Public Health Association American Water Works Association Water Environment Federation joint Editorial Board Arnold E. Greenberg, APHA, Chairman Lenore S. Qesceri, WEF Andrew D. Eaton, AWWA Managing Editor Mary Ann H. Franson Publication Office American Public Health Association 1015 Fifteenth Street, NW Washington, DC 20005 Copyright © 1917, 1920, 1923, and 1925 by American Public Health Association Copyright & 1933, 1936, and 1946 by American Public Health Association American Water Works Association Copyright © 1955, 1960, and 1965 by American Public Health Association American Water Works Association Water Pollution Control Federation Copyright ° 1971 by American Public Health Association American Water Works Association Water Pollution Control Federation Copyright® 1976 by American Public Health Association American Water Works Association Water Pollution Control Federation Copyright © 1 981 by American Public Health Association American Water Works Association Water Pollution Control Federation Copyright & 1985 by American Public Health Association American Water Works Association Water Pollution Control Federation Copyright © 1989 by American Public Health Association American Water Works Association Water Pollution Control Federation Copyright ° 1992 by A m erican Pub lie Health Association American Water Works Association Water Environment Federation All rights reserved. No part of this publication may be reproduced, graphically or electron- ically" including entering in storage or retrieval systems, without the prior written permission of the publishers. 3UM7/92 The Library of Congress has catalogued this work as follows: American Public Health Association. Standard methods for the examination of water and wastewater. ISBN 0-87553-207-1 Printed and bound in the United States of America. Composition: EPS Group, Inc., Hanover, Maryland Set in: Times Roman Printing: Victor Graphics, Inc., Baltimore, Maryland Binding: American Trade Bindery, Baltimore, Maryland Cover Design: DR Pollard and Associates, Inc., Arlington, Virginia 9-32 MICROBIOLOGICAL EXAMINATION (9000) 9215 HETEROTROPHIC PLATE COUNT* 9215 A. Introduction 1. Applications The heterotrophic plate count (HPC), formerly known as the standard plate count, is a procedure for estimating the number of live heterotrophic bacteria in water and measuring changes during water treatment and distribution or in swimming pools. Colonies may arise from pairs, chains, clusters, or single cells, all of which are included in the term '^colony-forming units" (CFU). The final count also depends on interaction among the developing colonies; choose that combination of procedure and medium that produces the greatest number of colonies within the designated incubation time. Three different methods and four different media are described. 2. Selection of Method guished readily from particles and bubbles. Colonies can be transferred quickly and compared easily to published descrip- tions. However, this method is limited by the small volume of sample or diluted sample that can be absorbed by the agar: 0.1 to 0.5 mL, depending on the degree to which the prepoured plates have been dried. To use this procedure, maintain a supply of suitable predried, absorbent agar plates. The membrane filter method (9215D) permits testing large volumes of low-turbidity water and is the method of choice for low-count waters (< 1 to 10 CFU/mL). This method produces no heat shock but adds the expense of the membrane filter. Further disadvantages include the smaller display area, the need to detect colonies by reflected light against a white background if colored filters or contrast stains are not used, and possible variations in membrane filter quality (see Section 9020B.3g). The pour plate method (9215B) is simple to perform and can accommodate volumes of sample or diluted sample ranging from 0.1 to 2.0 mL. The colonies produced are relatively small and compact, showing less tendency to encroach on each other than those produced by surface growth. On the other hand, sub- merged colonies often are slower growing, are difficult to trans- fer, and are not described in published studies. A thermostati- cally controlled water bath is essential for tempering the agar, but even so, significant heat shock from the transient exposure of the sample to 45 to 46°C agar may occur. The spread plate method (9215C) causes no heat shock and all colonies are on the agar surface where they can be distin- : Approved by Standard Methods Committee, 1988. 3. Work Area Provide a level table or bench top with ample area in a clean, draft-free, well-lighted room or within a horizontal-flow laminar hood. Use table and bench tops having nonporous surfaces and disinfect before any analysis is made. 4. Samples Collect water as directed in Section 9060A. Initiate analysis as soon as possible after collection to minimize changes in bac- terial population. The recommended maximum elapsed time be- tween collection and examination of samples is 8 h (maximum transit time 6 h, maximum processing time 2 h). When analysis cannot begin within 8 h, maintain sample at a temperature below HETEROTROPHIC PLATE COUNT (9215)/lntroduction 9-33 4°C but do not freeze. Do not let maximum elapsed time between collection and analysis exceed 24 h. Hold or transport bottled water samples obtained from retail outlets unrefrigerated under the same ambient conditions as those found in the retail store until tested. Examine freshly bottled samples (less than 48 h old) within 6 h of collection if unre- frigerated and within 24 h if refrigerated. 5. Sample Preparation Mark each plate with sample number, dilution, date, and any other necessary information before examination. Prepare at least duplicate plates for each volume of sample or dilution examined. For the pour or spread plate methods use glass (65 cm 2 ) or disposable plastic (57 cm 2 ) petri dishes. Thoroughly mix all samples or dilutions by rapidly making about 25 complete up-and-down (or back-and-forth) movements. Optionally, use a mechanical shaker to shake samples or dilutions for \5 s. 6. Media Compare new lots of media with current lot in use according to Section 9020B.3/z. a. Plate count agar (tryptone glucose yeast agar): Use for pour and spread plate methods. This high-nutrient agar, widely used in the past, gives lower counts than R2A or NWRI agar. It is included for laboratories wishing to make media comparisons or to extend the continuity of old data. Tryptone 5.0 g Yeast extract 2.5 g Glucose 1.0 g Agar 15.0 g Reagent-grade water 1 L pH should be 7.0 ± 0.2 after autoclaving at 121°C for 15 min. b. m-HPC agarr\ Use this high-nutrient medium only for the membrane filter method. Peptone 20.0 g Gelatin 25.0 g Glycerol 10.0 mL Agar 15.0 g Reagent-grade water 1 L Mix all ingredients except glycerol. Adjust pH to 7.1, if nec- essary, with \N NaOH, heat to dissolve, add glycerol, and au- toclave at 121°C for 5 min. c. R2A agar; Use for pour, spread plate, and membrane filter methods. This low-nutrient agar gives higher counts than high- nutrient formulations. Yeast extract 0.5 g Proteose peptone No. 3 or polypeptone 0.5 g Casamino acids 0.5 g Glucose 0.5 g Soluble starch 0.5 g Dipotassium hydrogen phosphate, K 2 HP0 4 0.3 g Formerly called m-SPC a car. Magnesium sulfate heptahydrate, MgS0 4 -7H 2 0.05 g Sodium pyruvate 0.3 g Agar 15.0 g Reagent-grade water 1 L Adjust pH to 7.2 with solid K 2 HP0 4 or KH 2 P0 4 before adding agar. Heat to dissolve agar and sterilize at 121°C for 15 min. d. NWRI agar (HPCA): Use for pour, spread plate, and mem- brane filter methods. This medium is likely to produce higher colony counts than the three media described above. It may not be available in dehydrated form and may require preparation from the basic ingredients. Peptone 3.0 g Soluble casein 0.5 g K 2 HP0 4 0.2 g MgSO, 0.05 g FeCI, 0.001 g Agar 15.0 g Reagent-grade water 1 L Adjust pH to 7.2 before autoclaving for 15 min at 121°C. 7. Incubation U.S. EPA revised regulations will describe the pour plate method. In testing to meet these regulations, incubate at 35°C for 48 h. Otherwise, select from among recommended times and temperatures for monitoring changes in water quality. The high- est counts typically will be obtained from 5- to 7-d incubation at a temperature of 20 to 28°C. If unable to provide incubators controlled at 20 to 28°C, use a dust-free, room-temperature cab- inet. Include time and temperature in reporting results. During incubation maintain humidity within the incubator so that plates will have no moisture weight loss greater than 15%. This is especially important if prolonged incubation is used. A pan of water placed at the bottom of the incubator may be sufficient but note that to prevent rusting or oxidation the inside walls and shelving should be of high-grade stainless steel or an- odized aluminum. For long incubation in nonhumidified incu- bators, seal plates in plastic bags. 8. Counting and Recording a. Pour and spread plates: Count all colonies on selected plates promptly after incubation. If counting must be delayed tempo- rarily, store plates at 5 to 10°C for no more than 24 h, but avoid this as routine practice. Record results of sterility controls on the report for each lot of samples. Use an approved counting aid, such as the Quebec colony counter, for manual counting. If such equipment is not available, count with any other counter provided that it gives equivalent magnification and illumination. Automatic plate counting in- struments are available. These generally use a television scanner coupled to a magnifying lens and an electronics package. Their use is acceptable if evaluation in parallel with manual counting gives comparable results. In preparing plates, plant sample volumes that will give from 30 to 300 colonies/plate. The aim is to have at least one dilution giving colony counts between these limits, except as provided below. 9-34 MICROBIOLOGICAL EXAMINATION (9000) Ordinarily, do not plant more than 2.0 mL of sample; there- fore, when the total number of colonies developing from 2.0 mL is less than 30, disregard the rule above and record result ob- served. With this exception, consider only plates having 30 to 300 colonies in determining the plate count. Compute bacterial count per milliliter by multiplying average number of colonies per plate by the reciprocal of the dilution used. Report counts as CFU per milliliter. If there is no plate with 30 to 300 colonies, and one or more plates have more than 300 colonies, use the plate(s) having a count nearest 300 colonies. Compute the count by multiplying average count per plate by the reciprocal of the dilution used and report as estimated CFU per milliliter. If plates from all dilutions of any sample have no colonies, report the count as less than one (< 1) times the reciprocal of the corresponding lowest dilution. For example, if no colonies develop on the 1:100 dilution, report the count as less than 100 (< 100) estimated CFU/mL. If the number of colonies per plate far exceeds 300, do not report result as "too numerous to count 1 ' (TNTC). If there are fewer than 10 colonies/cm 2 , count colonies in 13 squares (of the colony counter) having representative colony distribution. If pos- sible, select seven consecutive squares horizontally across the plate and six consecutive squares vertically, being careful not to count a square more than once. Multiply sum of the number of colonies in 13 representative square centimeters by 5 to compute estimated colonies per plate when the plate area is 65 cm 2 . When there are more than 10 colonies /cm 2 , count four representative squares, take average count per square centimeter, and multiply by the appropriate factor to estimate colonies per plate. The factor is 57 for disposable plastic plates and 65 for glass plates. When bacterial counts on crowded plates are greater than 100 colonies/cm 2 , report result as greater than (>) 6500 times the reciprocal of the highest dilution plated for glass plates or greater than (>) 5700 times the reciprocal for plastic plates. Report as estimated colony-forming units per milliliter. If spreading colonies (spreaders) are encountered on the plate(s) selected, count colonies on representative portions only when colonies are well distributed in spreader-free areas and the area covered by the spreader(s) does not exceed one-half the plate area. When spreading colonies must be counted, count each of the following types as one: a chain of colonies that appears to be caused by disintegration of a bacterial clump as agar and sample were mixed; a spreader that develops as a film of growth between the agar and bottom of petri dish; and a colony that forms in a film of water at the edge or over the agar surface. The last two types largely develop because of an accumulation of moisture at the point from which the spreader originates. They frequently cover more than half the plate and interfere with obtaining a reliable plate count. Count as individual colonies similar-appearing colonies grow- ing in close proximity but not touching, provided that the distance between them is at least equal to the diameter of the smallest colony. Count impinging colonies that differ in appearance, such as morphology or color, as individual colonies. If plates have excessive spreader growth, report as "spreaders" (Spr). When plates are uncountable because of missed dilution, accidental dropping, and contamination, or the control plates indicate that the medium or other material or labware was con- taminated, report as "laboratory accident' 1 (LA). b. Membrane filter method: Count colonies on membrane fil- ters using a stereoscopic microscope at 10 to 15 x magnification. Preferably place petri dish on microscope stage slanted at 45° and adjust light source vertical to the colonies. Optimal colony density per filter is 20 to 200. If colonies are small and there is no crowding, a higher limit is acceptable. Count all colonies on the membrane when there are 1 to 2, or fewer, colonies per square. For 3 to 10 colonies per square count 10 squares and obtain average count per square. For 10 to 20 colonies per square count 5 squares and obtain average count per square. Multiply average count per square by 100 times the reciprocal of the dilution to give colonies per milliliter. If there are more than 20 colonies per square, record count as > 2000 times the reciprocal of the dilution. Report averaged counts as estimated colony-forming units. Make estimated counts only when there are discrete, separated colonies without spread- ers. 9. Computing and Reporting Counts The term "colony-forming units' 1 (CFU) is descriptive of the methods used; therefore, report all counts as colony-forming units. Include in the report the method used, the incubation temperature and time, and the medium. For example: CFU/mL, pour plate method, 35°C/48 h, plate count agar or 28°/5 d, R2A agar; or CFU/mL, spread plate method, 2077 d, NWRI agar. To compute the heterotrophic plate count, CFU/mL, multiply total number of colonies or average number (if duplicate plates of the same dilution) per plate by the reciprocal of the dilution used. Record dilutions used and number of colonies on each plate counted or estimated. When colonies on duplicate plates and/or consecutive dilutions are counted and results are averaged before being recorded, round off counts to two significant figures only when converting to colony-forming units. Avoid creating fictitious precision and accuracy when com- puting colony-forming units by recording only the first two left- hand digits. Raise the second digit to the next higher number when the third digit from the left is 5, 6, 7, 8, or 9; use zeros for each successive digit toward the right from the second digit. For example, report a count of 142 as 140 and a count of 155 as 160, but report a count of 35 as 35. 10. Personal Errors Avoid inaccuracies in counting due to carelessness, damaged or dirty optics that impair vision, or failure to recognize colonies. Laboratory workers who cannot duplicate their own counts on the same plate within 5% and the counts of other analysts within 10% should discover the cause and correct such disagreements. HETEROTROPHIC PLATE COUNT (9215)/Pour Plate Method 9215 B. Pour Plate Method 9-35 1 . Samples and Sample Preparation See 9215A.4 and 9215A.5. 2. Sample Dilution Prepare water used for dilution blanks as directed in Section 9050C. a. Selecting dilutions: Select the dilution(s) so that the total number of colonies on a plate will be between 30 and 300 (Figure 9215:1). For example, where a heterotrophic plate count as high as 3000 is suspected, prepare plates with 10~ 2 dilution. For most potable water samples, plates suitable for counting will be obtained by plating 1 mL and 0.1 mL undiluted sample and 1 mL of the 10 2 dilution. b. Measuring sample portions: Use a sterile pipet for initial and subsequent transfers from each container. If pipet becomes contaminated before transfers are completed, replace with a ster- ile pipet. Use a separate sterile pipet for transfers from each different dilution. Do not prepare dilutions and pour plates in direct sunlight. Use caution when removing sterile pipets from the container; to avoid contamination, do not drag pipet tip across exposed ends of pipets in the pipet container or across lips and necks of dilution bottles. When removing sample, do not insert pipets more than 2.5 cm below the surface of sample or dilution. c. Measuring dilutions: When discharging sample portions, hold pipet at an angle of about 45° with tip touching bottom of petri dish or inside neck of dilution bottle. Lift cover of petri dish just high enough to insert pipet. Allow 2 to 4 s for liquid to drain from 1-mL graduation mark to tip of pipet. If pipet is not a blow- out type, touch tip of pipet once against a dry spot on petri dish bottom. Less preferably, use a cotton-plugged blow-out-type pi- pet and gently blow out remaining volume of sample dilution. When 0.1-mL quantities are measured, let diluted sample drain from chosen reference graduation until 0.1 mL has been deliv- ered. Remove pipet without retouching it to dish. Pipet 1 mL, 0.1 mL, or other suitable volume into sterile petri dish before adding melted culture medium. Use decimal dilutions in pre- paring sample volumes of less than 0.1 mL; in examining sewage or turbid water, do not measure a 0.1-mL inoculum of original sample, but prepare an appropriate dilution. Prepare at least two replicate plates for each sample dilution used. After depositing test portions for each series of plates, pour culture medium and mix carefully. Do not let more than 20 min elapse between start- ing pipetting and pouring plates. 3. Plating a. Melting medium: Melt sterile solid agar medium in boiling water or by exposure to flowing steam in a partially closed con- tainer, but avoid prolonged exposure to unnecessarily high tem- peratures during and after melting. Do not resterilize plating medium. If the medium is melted in two or more batches, use all of each batch in order of melting, provided that the contents remain fully melted. Discard melted agar that contains precipi- tate. Maintain melted medium in a water bath between 44 and 46°C until used. In a separate container place a thermometer in water or medium that has been exposed to the same heating and cooling as the plating medium. Do not depend on the sense of touch to indicate proper medium temperature when pouring agar. Use plate count agar, R2A agar, or NWRI agar as specified in Section 9215A.6. Before using a new lot of medium test its suitability. Delivery volume Culture dishes Actual volume oi' sample in dish 1 mL 0.1 mL 10" 2 mL Figure 9215:1. Preparation of dilutions. 10 ; ' mL 9-36 MICROBIOLOGICAL EXAMINATION (9000) b. Pouring plates: Limit the number of samples to be plated in any one series so that no more than 20 min (preferably 10 min) elapse between dilution of the first sample and pouring of the last plate in the series. Pour at least 10 to 12 mL liquefied medium maintained at 44 to 46°C into each dish by gently lifting cover just high enough to pour. Carefully avoid spilling medium on outside of container or on inside of dish lid when pouring. As each plate is poured mix melted medium thoroughly with test portions in petri dish, taking care not to splash mixture over the edge, by rotating the dish first in one direction and then in the opposite direction, or by rotating and tilting. Let plates solidify (within 10 min) on a level surface. After medium solidifies, invert plates and place in incubator. c. Sterility controls: Check sterility of medium and dilution water blanks by pouring control plates for each series of samples. Prepare additional controls to determine contamination of plates, pipets, and room air. 4. Incubation See Section 9215A.7. 5. Counting, Recording, Computing, and Reporting See Sections 9215A.8 and 9215A.9. 6. Bibliography Breed, R.S. & W.D. Dotterer. 1916. The number of colonies allow- able on satisfactory agar plates. Tech. Bull. 53, New York Agri- cultural Experiment Sta. Butterfield, C.T. 1933. The selection of a dilution water for bacte- riological examinations. J. BacterioL 23:355; Pub. Health Rep. 48:681. Archambault, J., J. Curot & M.H. McCrady. 1937. The need of uniformity of conditions for counting plates (with suggestions for a standard colony counter). Amer. J. Pub. Health 27:809. Richards, O.W. & P.C. Heijn. 1945. An improved dark- field Quebec colony counter. J. Milk Tech not. 8:253. Berry, J.M., D.A. McNeill & L.D. Witter. 1969. Effect of delays in pour plating on bacterial counts. J. Dairy Sci. 52:1456. Geldreich, E.E., H.D. Nash, D.J. Reasoner& R.H. Taylor. 1972. The necessity of controlling bacterial populations in potable waters: Community water supply. J. Amer. Water Works Assoc. 64:596. Geldreich, E.E. 1973. fs the total count necessary? Proc. 1st Annu. Water Quality Teehnol. Conf. , American Water Works Assoc, Pa- per No. VIM. Ginsburg, W. 1973. Improved total count techniques. Proc. 1st Annu. Water Quality Teehnol. Conf., American Water Works Assoc, Pa- per No. VTIL Dutka, B.J., A.S.Y. Chau & J. Coburn. 1974. Relationship of het- erotrophic bacterial indicators of water pollution and fecal sterols. Water Res. 8:1047. Klein, D.A. & S. Wu. 1974. Stress: a factor to be considered in het- erotrophic microorganism enumeration from aquatic environments. Appl. Microbiol. 37:429. Geldreich, E.E. , H.D. Nash, D.J. Reasoner & R.H. Taylor. 1975. The necessity for controlling bacterial populations in potable waters: Bottled water and emergency water supplies. ,/. Amer. Water Works Assoc. 67:117. Bell, C.R., M.A. Holder-Franklin & M. Franklin. 1980. Heter- otrophic bacteria in two Canadian rivers. — I. Seasonal variation in the predominant bacterial populations. Water Res. 14:449. Reasoner, D.J. & E.E. Geldreich. 1981. fn flue nee of medium, meth- ods and incubation time and temperature on the bacterial count of potable water. 8 1st Annu. Meeting, American Soc. Microbiology, Dallas, Tex., Paper No. N27. Means, E.G., L. Hanami, G.F. Ridgway & B.H. Olson. 1981. Eval- uating mediums and plating techniques for enumerating bacteria in water distribution systems. J. Amer. Water Works Assoc. 73:585. American Public Health Association. 1985. Standard Methods for the Examination of Dairy Products, 15th ed. American Public Health Assoc, Washington, D,C. Reasoner, D.J. & E.E. Geldreich. 1985. A new medium for the enumeration and subculture of bacteria from potable water. Appl. Environ. Microbiol. 49:1. 9215 C. Spread Plate Method 1 . Laboratory Apparatus a. Glass rods: Bend 4-mm-diam fire-polished glass rods, 200 mm in length, 45° about 40 mm from one end. Sterilize before using. b. Pipet, glass, 1.1 mL, with tempered, rounded tip. Do not use disposable plastic pipets. c. Turntable (optional).* d. Incubator or drying oven, set at 42°C, or laminar-flow hood. 2. Media 3. Preparation of Plates Pour 15 mL of the desired medium into sterile 100 x 15 or 90 x 15 petri dishes; let agar solidify. Predry plates inverted so that there is a 2- to 3-g water loss overnight with lids on. See Figure 9215:2, Table 9215:1, or Figure 9215:3. Use predried plates immediately after drying. For predrying and using plates the same day, pour 25 mL agar into petri dish and dry in a laminar- flow hood at room temperature (24 to 26°C) with the lid off to obtain the desired 2- to 3-g weight loss. See Figure 9215:3. See 92 15 A. 6a, c, and d. If R2A agar is used best results are obtained at 28°C with 7 d incubation; if NWRI is used, incubate at 20°C for 7 d. * Fisher Scientific, hand operated, No. 08-758 or Lab- Line motor driven. No. 1580, or equivalent. 4. Procedure Prepare sample dilutions as directed in 921 5B. 2. a. Glass rod: Pipet 0.1 or 0.5 mL sample onto surface of predried agar plate. Using a sterile bent glass rod, distribute inoculum over surface of the medium by rotating the dish by HETEROTROPHIC PLATE COUNT (9215)/Spread Plate Method 9-37 y/50"C "•''42* C Figure 9215:2. Drying weight loss of 15-mL agar plates stored separately, inverted with lids on. Source: Unpublished data. Water Purification Lab., Chicago Dep. Water. O Near screen S Near screen. 1 2 lids oft a Near ouiside edge fids off O Near outside edge i 2 lids off A Random placement lids off Figure 9215:3. Weight loss of 25-mL agar plates (100 X 15 mm) dried separately in a laminar-flow hood at room temperature (24 to 26°C), relative humidity (30 to 33%), and air velocity 0.6 m/s. Source: Unpublished data. Alberta Environmental Centre, Vengreville, Alta. 9-38 MICROBIOLOGICAL EXAMINATION (9000) Ta b l n 9215:1. Effect o i ■' Te m p e r atu r f o f D r y inc; on W e i o. ht Loss of 15-mL Agar Plates Six) red Separately* rime for Plates to Lose 1 to 4 g of Water (Avg. for 5 Plates) h Plates Inverted Plates I nverted Temp. °C with Lids On with Lids Removed I g 2 £ 3 g 4 g lg 9 a 3g 4g 24 32 64 95 125 3.7 7.0 10.5 14.0 37 17 35 51 67 1.7 3.5 5.3 7.0 50 6 12 18 24 0.7 1.3 1.9 2.7 60 4 8 12 16 __ — — — * Referenced in Canada Centre for Inland Waters Manual. Burl inelon. Out. hand or on a turntable. Let inoculum be absorbed completely into the medium before incubating. b. Piper : Pipet desired sample volume (0.1, 0.5 mL) onto the surface of the predried agar plate while dish is being rotated on a turntable. Slowly release sample from pipet while making one to-and-fro motion, starting at center of the plate and stopping 0.5 cm from the plate edge before returning to the center. Lightly touch the pipet to the plate surface. Let inoculum be absorbed completely by the medium before incubating. 5. Incubation See 9215A.7. 6. Counting, Recording, Computing, and Reporting See 9215A.8 and 9215A.9. 7. Bibliography Buck, J.D. & R.C. Clever don. 1960. The spread plate as a method for the enumeration of marine bacteria. Umnol. Occanogr. 5:78. Clark, D.S. 1967. Comparison of pour and surface plate methods for determination of bacterial counts. Can. J. Microbiol. 13:1409. Van Soestbergan, A. A. & C.H. Lee. 1969. Pour plates or streak plates. Appi. Microbiol. 18:1092. Clark, D.S. 1971. Studies on the surface plate method of counting bacteria. Can. J. Microbiol J 7:943. Gilchrist, J.E., J.E. Campbell, C.B. Donnelly, J.T. Peeler & J.M. Delaney. 1973. Spiral plate method for bacterial determi- nation. Appi. Microbiol. 25:244. Ptak, D.M. & W. Ginsburg. 1976. Pour plate vs. streak plate method. Proc. 4th Annu. Water Quality Teehnol. Conf., American Water Works Assoc, Paper No. 2B-5. Dutka, B.J., ed. 1978. Methods for Microbiological Analysis of Waters, Wastewaters and Sediments. Inland Waters Directorate, Scientific Operation Div., Canada Centre for Inland Waters, Burlington, On t. Kaper, J.B., A.L. Mills & R.R. Colwell. 1978. Evaluation of the accuracy and precision of enumerating aerobic heterotrophs in water samples by the spread method. Appi. Environ. Microbiol. 35:756. Young, M. 1979. A modified spread plate technique for the determi- nation of concentrations of viable heterotrophic bacteria. STP 673:41- 51, American Soc. Testing & Materials. Philadelphia, Pa. Geldreich, E.E. 1981. Current status of microbiological water quality criteria. ASM News 47:23. Taylor, R.H., M..L Allen & E.E. Geldreich. 1981. Standard plate count: A comparison of pour plate and spread plate methods. Proc. 9th Annu. Water Quality Teehnol. Conf., American Water Works Assoc. 9215 D. Membrane Filter Method 1 . Laboratory Apparatus See Section 9222B.1. 2. Media See 9215A.6. Use m-HPC agar, or alternatively R2A or NWRI agar. 3. Preparation of Plates Dispense 5-mL portions of sterile medium into 50- x 9-mm petri dishes. Let solidify at room temperature. Prepared plates may be stored inverted in a plastic bag or tight container in a refrigerator, preferably for no longer than 1 week. 5. Procedure Filter appropriate volume through a sterile 47-mm, 0.45-fim, gridded membrane filter, under partial vacuum. Rinse funnel with three 20- to 30-mL portions of sterile dilution water. Place filter on agar in petri dish. 6. Incubation Place dishes in close fitting box or plastic bag containing mois- tened paper towels. Incubate at 35 ± 0.5°C for 48 h if using m- HPC agar, or longer if using R2A medium, or at 20°C for 7d if using NWRI agar. Duplicate plates may be incubated for other time and temperature conditions as desired. 4. Sample Size The volume to be filtered will vary with the sample. Select a maximum sample size to give 20 to 200 CFU per filter. 7. Counting, Recording, Computing, and Reporting See 9215A.8 and 9215A.9. Report as CFU/mL, membrane filter method, time, medium. DIRECT TOTAL COUNT (9216)/Epifluorescence Method 9-39 8. Bibliography Clark, H.F., E.E. Geldreich, H.L. Jeter Sc P.W. Kabler. 1951. The membrane filler in sanitary bacteriology. Pub. Health Rep. 66:951. Stopert, E.M., W.T. Sokoski & J.T. Northam. 1962. The factor of temperature in the better recovery of bacteria from water by nitra- tion. Can. ./. Microbiol. 8:809. Taylor, R.H. & E.E. Geldreich. 1979. A new membrane filter pro- cedure for bacterial counts in potable water and swimming pool samples. J. A frier. Water Works Assoc. 71:402. Clark, J. A. 1980. The influence of increasing numbers of non-indicator organisms upon the detection of indicator organisms by the mem- brane filter and presence-absence tests. Can. J. Microbiol. 20:827. Dutka, B.J., ed. 1981 . Membrane Filtration, Applications, Techniques, and Problems. Marcel Dekker, Inc., New York, N.Y. and Basel. Switzerland. Hoadley, A.W. 1981. Effect of injury on the recovery of bacteria on membrane filters. In B. J. Dutka, ed. Membrane Filtration, Ap- plications, Techniques, and Problems, pp. 413-450. Marcel Dekker, Inc., New York. N.Y. and Basel, Switzerland. d. Blender or vortex mixer. : Bausch & Lomb No. 3 1-16- .13. ■ K. Zeiss or equivalent. ± Nuclepore Corp. or equivalent. § Milliporc Corp. or equivalent. jj Sigma Chemical Co., biological grade, or equivalent. # Cargillc Laboratories, Inc., Type B. or equivalent.